CN115210574A

CN115210574A - High confidence compound identification by liquid chromatography-mass spectrometry

Info

Publication number: CN115210574A
Application number: CN202180011554.XA
Authority: CN
Inventors: 吴骥康; 王红霞; 邱海波; 李宁
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2020-01-31
Filing date: 2021-01-29
Publication date: 2022-10-18
Also published as: MX2022009411A; BR112022014901A2; EP4097483A1; JP2023512069A; CA3166241A1; US20220365045A1; AU2021212211A1; IL294907A; WO2021155219A1; US20210239660A1; KR20220134585A; US11371971B2; US11959897B2

Abstract

Methods for improving compound detection and characterization are disclosed. Methods of characterizing a sample are disclosed. The method may include providing a sample to a liquid chromatography system capable of sample separation to generate sample components; analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list; and performing iterative mass spectrometry Data Dependent Acquisition (DDA) from the inclusion list to identify individual sample components to characterize the sample. In one example, multiplexed targeted SIM and iterative MS2 DDA acquisitions are used to increase robust compound identification for cell culture medium analysis.

Description

High confidence compound identification by liquid chromatography-mass spectrometry

Technical Field

The invention relates to compound identification, in particular to a method for identifying a high-confidence compound by liquid chromatography-mass spectrometry (LC-MS), such as antibody process development.

Background

Cell culture media plays a key role in antibody production. It is highly desirable and challenging to understand how individual components and their metabolites in cell culture media affect production performance. Despite the change in the use of chemically defined media, soy-based media is still widely used. Major components and impurities of soy hydrolysate have been shown to affect the productivity and quality of antibodies. Mass spectrometry plays an important role in the quantification and identification of compounds in highly complex matrices. Liquid chromatography-mass spectrometry (LC-MS) based analysis can greatly benefit from improved data quality, which can then be characterized with higher confidence and lower ambiguity.

Disclosure of Invention

In one aspect, the present invention provides a method of characterizing a sample, comprising: providing the sample to a liquid chromatography system capable of sample separation to generate sample components; analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list; and performing iterative mass spectrometry Data Dependent Acquisition (DDA) from the inclusion list to identify individual sample components to characterize the sample.

In some embodiments, the liquid chromatography system is a Reverse Phase Liquid Chromatography (RPLC) system.

In some embodiments, analyzing the ionized sample by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list comprises utilizing an ion trap or an orbitrap mass analyzer.

In some embodiments, performing iterative mass spectrometry Data Dependent Acquisition (DDA) includes utilizing an ion trap or an orbitrap mass analyzer equipped with a segmented quadrupole mass filter.

In some embodiments, analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list comprises a segmented mass ratio window set, wherein a plurality of segments are included and each segment has a plurality of windows.

In some embodiments, the plurality of segments is three segments.

In some embodiments, the plurality of segments is four segments.

In some embodiments, the plurality of windows is 10 windows.

In some embodiments, each window within a segment has the same window width.

In some embodiments, the sample is a cell culture medium.

In some embodiments, the cell culture medium is a soy-based cell culture medium.

In some embodiments, the cell culture medium is used in a recombinant cell-based production system.

In some embodiments, the method is used to characterize components in cell culture media and metabolites thereof after incubation with recombinant cell-based production systems.

In some embodiments, the recombinant cell-based production system is a mammalian system.

In some embodiments, recombinant cell-based production systems are used for protein production.

In some embodiments, the protein is an antibody, a fusion protein, a recombinant protein, or a combination thereof.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the monoclonal antibody is of isotype IgG1, igG2, igG3, igG4 or mixed isotypes.

Also disclosed is a method of compound identification for cell culture medium analysis comprising: providing a cell culture medium sample to a liquid chromatography system capable of sample separation to generate sample components; analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list; and performing iterative mass spectrometry Data Dependent Acquisition (DDA) from the inclusion list to identify individual compounds in the cell culture medium.

In some embodiments, analyzing the fragmented sample by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list comprises utilizing an ion trap or orbitrap mass analyzer.

In some embodiments, the plurality of segments is three segments.

In some embodiments, the plurality of segments is four segments.

In some embodiments, the plurality of windows is 10 windows.

In some embodiments, each window within a segment has the same window width.

In some embodiments, the cell culture medium sample is a cell culture medium sample obtained after incubation with a recombinant cell-based production system.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the monoclonal antibody is of isotype IgG1, igG2, igG3, igG4, or mixed isotypes.

In various embodiments, any of the features or components of the embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any particular value discussed above or herein can be combined with another related value discussed above or herein to recite a range, wherein the value represents an upper and lower limit of the range, and such range and all values falling within such range are encompassed within the scope of the present disclosure. Each of the values discussed above or herein may be expressed as a 1%, 5%, 10%, or 20% variation. Other embodiments will become apparent from a reading of the following detailed description.

Drawings

Fig. 1 shows a schematic diagram illustrating two types of MS1 data acquisition according to embodiments disclosed herein: (1) Conventional full scan MS1 acquisition and (2) targeted SIM MS1 acquisition. As shown, targeted SIM MS1 acquisition allowed detection of lower abundance species.

Fig. 2 shows a schematic diagram illustrating an exemplary adjustable window selection protocol.

Fig. 3 shows MS1 spectra acquired after a conventional full scan MS1 acquisition (top panel) or an adjustable window SIM MS1 acquisition (bottom panel) according to embodiments disclosed herein.

Fig. 4 shows a schematic diagram illustrating two types of MS2 DDA, according to embodiments disclosed herein: (1) Conventional DDA MS2 acquisition and (2) iterative MS2 DDA platform acquisition.

Figure 5 shows the iterative MS2 DDA results of selecting and fragmenting more species than conventional MS2 DDA of only selecting and fragmenting the high abundance species.

Fig. 6 shows a schematic diagram of a conventional method of comparative characterization of samples and a combination of acquisition and iterative MS2 DDA using an adjustable window SIM according to embodiments disclosed herein.

Fig. 7 shows a table illustrating the robustness and increased sensitivity of adjustable window SIM acquisition compared to a full scan MS. The ability to detect a particular molecule as the concentration of the compound decreases depends on the method used. The adjustable window SIM acquisition is able to detect molecules at lower concentrations than full scan MS.

Fig. 8 shows a graph illustrating the intensity of an iterative MS2 DDA. The top row represents the number of entries on the filter exclusion list, while the bottom row is the number of entries on the filter inclusion list, each entry associated with an iterated sample injection. As shown, as the number of iterative samplings increases, the exclusion list of species increases, while the inclusion list decreases, which in turn allows lower abundance species to be fragmented and provides a robust, high sensitivity detection method.

Detailed Description

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Any embodiments or features of embodiments may be combined with each other and such combinations are expressly contemplated within the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a specifically recited value, means that the value may differ from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "comprising," "including," and "including" are intended to be non-limiting and should be understood to mean "comprising," "comprises," and "comprising," respectively.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patent, application, and non-patent publications mentioned in this specification are herein incorporated by reference in their entirety.

Abbreviations used herein

ACN: acetonitrile

CHO: chinese hamster ovary

CQA: key quality attribute

CV: coefficient of variation

DDA: data dependent acquisition

EIC: extracted ion chromatograph

ESI-MS: electrospray ionization mass spectrometry

And (2) FA: formic acid

HC: heavy chain

HILIC: hydrophilic interaction liquid chromatography

HMW: high molecular weight

IgG: immunoglobulin G

IPA: isopropanol (I-propanol)

LC: light chain

LC-MS: liquid chromatography-mass spectrometry

LMW: low molecular weight

mAb: monoclonal antibodies

And (2) MS: mass spectrometry

MW: molecular weight

M/Z: mass to charge ratio

NCE: normalized collision energy

PK: pharmacokinetics

PQA: product quality attributes

And (4) PTM: post-translational modification

RP-LC: reversed phase liquid chromatography

SIM: selective ion monitoring

Definition of

As used herein, the term "protein" includes any polymer of amino acids having covalently linked amide bonds. Proteins comprise one or more polymer chains of amino acids, commonly referred to in the art as "polypeptides". "polypeptide" refers to a polymer composed of amino acid residues, their related naturally occurring structural variants and synthetic non-naturally occurring analogs linked by peptide bonds. "synthetic peptide or polypeptide" refers to a peptide or polypeptide that does not occur in nature. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those skilled in the art. A protein may contain one or more polypeptides to form a single functional biomolecule. The protein may include any biotherapeutic protein, recombinant proteins for research or therapy, trap proteins and other chimeric receptor Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In another exemplary aspect, the protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives, such as CHO-K1 cells), can be used to produce proteins. For a recent review on the discussion of biotherapeutic proteins and their production, see Ghaderi et al, a platform for the production of biotherapeutic glycoproteins. Development, impact and challenge of non-human sialylation (Production plants for biological glycopeptides, impact, and galleries of non-human sialylation) (review of biotechnology and genetic engineering (Biotechnol. Genet. Eng. Rev.) (2012) 147-75). In some embodiments, the protein comprises modifications, adducts, and other covalently linked moieties. Such modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose Binding Protein (MBP), chitin Binding Protein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescent labels and other dyes, and the like. Proteins may be classified based on composition and solubility, and thus may include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derivatized proteins, such as primary and secondary derivatized proteins.

As used herein, a "variant protein" or "protein variant" or "variant" may include a protein that differs from a target protein by at least one amino acid modification. A protein variant may refer to the protein itself, a composition comprising the protein, or an amino sequence encoding it. Preferably, the protein variant has at least one amino acid modification as compared to the parent protein, for example from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications as compared to the parent. The protein variant sequences herein preferably have at least about 80% homology, and most preferably at least about 90% homology, more preferably at least about 95% homology to the parent protein sequence. In some exemplary embodiments, the protein can be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, or a combination thereof.

The term "antibody" as used herein refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) and two light (L) chains linked to each other by disulfide bonds (i.e., a "whole antibody molecule"), as well as multimers thereof (e.g., igM) or antigen-binding fragments thereof. Each heavy chain is composed of a heavy chain variable region ("HCVR" or "V _H ") and heavy chain constant region (consisting of Domain C _H 1，C _H 2 and C _H 3) is prepared. In various embodiments, the heavy chain can be of an IgG isotype. In some cases, the heavy chain is selected from IgG1, igG2, igG3, or IgG4. In some embodiments, the heavy chain is of isotype IgG1 or IgG4, optionally including a chimeric hinge region of isotype IgG1/IgG2 or IgG4/IgG 2. Each light chain is composed of a light chain variable region ("LCVR or" V _L ") and a light chain constant region (C) _L ) And (4) forming. V _H Region and V _L The regions may be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs) interspersed with more conserved regions known as Framework Regions (FRs). Each V _H And V _L Consisting of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody. For an overview of antibody structure, see Lefranc et al, IMGT unique numbers for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains (IMGT unique number for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains), 27 (1) development and comparison immunology (dev. Comp. Immunol.) 55-77 (2003); potter, structural correlates of immunoglobulin diversity (Structural proteins of immunoglobulin diversity), 2 (1) immunology studies of survivin (surv. Immunol.) Res.) -27-42 (1983).

The term antibody also includes "bispecific antibodies," which include heterotetrameric immunoglobulins that can bind more than one different epitope. Half of a bispecific antibody comprising a single heavy chain and a single light chain and six CDRs binds to one antigen or epitope, and the other half binds to a different antigen or epitope. In some cases, bispecific antibodies may bind to the same antigen, but at different epitopes or non-overlapping epitopes. In some cases, the bispecific antibodies of both halves have the same light chain while maintaining dual specificity. Bispecific antibodies are generally described in U.S. patent application publication No. 2010/0331527 (12/30/2010).

The term "antigen-binding portion" of an antibody (or "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al (1989) Nature 241-544 to 546) consisting of VH domains, (vi) isolated CDRs, and (vii) scFv consisting of the two domains VL and VH of the Fv fragment, joined by a synthetic linker to form a single protein chain, in which the VL and VH regions pair to form monovalent molecules. The term "antibody" also encompasses other forms of single chain antibodies, such as diabodies (see, e.g., holliger et al (1993) 90, proc. Natl. Acad. Sci. USA (PNAS U.S. A.), "6444-6448; and Poljak et al (1994) 2, structure (Structure) 1121-1123).

In addition, antibodies and antigen-binding fragments thereof can be obtained using standard recombinant DNA techniques commonly known in the art (see Sambrook et al, 1989). Methods for making human antibodies in transgenic mice are also known in the art. For example, use

Techniques (see, e.g., U.S. Pat. No. 6,596,541, regeneron Pharmaceuticals),

) Or any other known method for generating monoclonal antibodies, a high affinity chimeric antibody having human variable regions and mouse constant regions is initially isolated against the desired antigen.

The technology relates to the production of a transgenic mouse having a genome comprising a human heavy chain variable region and a human light chain variable region operably linked to an endogenous mouse constant region locus such that the mouse produces antibodies comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the heavy chain variable region and the light chain variable region of the antibody is isolated and operably linked to DNA encoding the human heavy chain constant region and the human light chain constant region. The DNA is then expressed in cells capable of expressing fully human antibodies.

The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mabs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, particularly in CDR 3. However, as used herein, the term "human antibody" is not intended to include mabs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammalian or non-human mammalian cells. The term is not intended to include antibodies isolated from or produced in a human subject.

As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, for example, in need of amelioration, prevention and/or treatment of a disease or disorder.

As used herein, the term "impurities" may include any unwanted proteins present in the biopharmaceutical product. The impurities may include impurities associated with the process and the product. Impurities may also be of known structure, partially characterized, or unidentified. Process related impurities may originate from the manufacturing process and may include three main categories: cell substrate sources, cell culture sources, and downstream sources. Impurities of cellular substrate origin include, but are not limited to, proteins and nucleic acids (host cell genome, vector or total DNA) derived from the host organism. Cell culture derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Impurities of downstream origin include, but are not limited to, enzymes, chemical and biochemical treatment reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metal ions), solvents, carriers, ligands (e.g., monoclonal antibodies) and other leachables. Product-related impurities (e.g., precursors, certain degradation products) can be molecular variants produced during manufacture and/or storage that do not have properties comparable to those of the desired product in terms of activity, efficacy, and safety. Such variants may require considerable effort in isolation and characterization to identify the type of modification. Product-related impurities may include truncated forms, modified forms, and aggregates. The truncated form is formed by a hydrolase or a chemical that catalyzes the cleavage of a peptide bond. Modified forms include, but are not limited to, deamidation, isomerization, mismatched S-S linkage, oxidized or altered conjugated forms (e.g., glycosylation, phosphorylation). Modified forms may also include any post-translational modification. Aggregates include dimers and higher multiples of the desired product (Q6B specification: biotechnology/Bioproduct test procedure and acceptance criteria, ICH 1999, 8.8.4.U.S. department of Health and human Services).

The term "Low Molecular Weight (LMW) protein drug impurities" includes, but is not limited to, precursors, degradation products, truncated species, proteolytic fragments including Fab fragments, fc or heavy chain fragments, ligand or receptor fragments, H2L (2 heavy chains and 1 light chain), H2 (2 heavy chains), HL (1 heavy chain and 1 light chain), HC (1 heavy chain), and LC (1 light chain) species. The LMW protein drug impurity may be any variant of an incomplete form of the protein product, such as one or more components of a multimeric protein. Protein drug impurities, drug impurities or product impurities are terms used interchangeably throughout the specification. LMW drug or product impurities are generally considered to be molecular variants with properties such as activity, efficacy, and safety that may differ from the desired drug product.

Degradation of protein products is problematic during the production of protein drug products in cell culture systems. For example, proteolysis of protein products may occur due to the release of proteases in the cell culture medium. Media additives, such as soluble iron sources added to inhibit metalloproteinases, or serine and cysteine protease inhibitors, have been implemented in cell culture to prevent degradation (Clincke, m. -f. Et al, "british conference of medical commission (BMC.) 2011,5, P115). The C-terminal fragment can be cleaved during production by carboxypeptidases in cell culture (Dick, LW et al, biotech and Bioengineering (Biotechnol Bioeng) 2008.

The term "High Molecular Weight (HMW) protein drug impurity" includes, but is not limited to, mAb trimers and mAb dimers. HMW species can be divided into two groups: 1) Monomers with additional light chains (H2L 3 and H2L4 species) and 2) monomer plus Fab fragment complexes. In addition, different dimerized fragments (Fab 2-Fab2, fc-Fc, and Fab 2-Fc) were formed after digestion with the IdeS enzyme.

"post-translational modification" (PTM) refers to covalent modification of a protein following its biosynthesis. Post-translational modifications can occur at the amino acid side chain or at the C-or N-terminus of the protein. PTMs are typically introduced by specific enzymes or enzymatic pathways. Many occur at sites within the protein backbone that are specific for a particular characteristic protein sequence (e.g., a signature sequence). Hundreds of PTMs have been recorded, and these modifications invariably affect certain aspects of protein structure or function (Walsh, g., proteins (Proteins), 2014, second edition, published by willey father publishing company (Wiley and Sons, ltd.), ISBN: 9780470669853). Various post-translational modifications include, but are not limited to, cleavage, N-terminal extension, protein degradation, acylation of the N-terminus, biotinylation (lysine residues are acylated with biotinylation), amidation of the C-terminus, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation (acetyl groups are typically added at the N-terminus of the protein), alkylation (alkyl groups are typically added at lysine or arginine residues (e.g., methyl, ethyl, propyl)), methylation, adenylation, ADP-ribosylation, covalent cross-linking within or between polypeptide chains, sulfonation, prenylation, vitamin C-dependent modifications (proline and lysine hydroxylation and carboxyl-terminal amidation), vitamin K-dependent modifications (where vitamin K is a cofactor in the carboxylation of glutamic acid residues, resulting in the formation of gamma-carboxyglutamic acid (glu residue)), glutamination (covalent attachment of glutamic acid residues), glycination (covalent attachment of glycine residues), glycosylation (addition of a sugar group to asparagine, hydroxylysine, serine or threonine to produce a glycoprotein), prenylation (addition of isoprenoid groups such as farnesol and geranylgeraniol), lipoylation (attachment of lipoic acid ester groups), phosphopantetheination (addition of a 4' -phosphopantetheinyl moiety from coenzyme A, such as in fatty acid, polyketide, non-ribosomal peptide and leucine biosynthesis), phosphorylation (addition of phosphate groups, typically to serine, threonine, tyrosine, and the like), and the like, tyrosine, threonine or histidine) and sulfation (addition of a sulfate group, typically to a tyrosine residue). Post-translational modifications that alter the chemistry of an amino acid include, but are not limited to, citrullination (e.g., arginine to citrulline by deimination) and deamidation (e.g., glutamine to glutamate or asparagine to aspartate). Post-translational modifications involving structural changes include, but are not limited to, the formation of disulfide bridges (covalent linkage of two cysteine amino acids) and proteolytic cleavage (cleavage of the protein at peptide bonds). Certain post-translational modifications involve the addition of other proteins or peptides, such as ISG acylation (covalent linkage to ISG15 protein (interferon stimulating gene)), SUMO acylation (covalent linkage to SUMO protein (small ubiquitin related modifier gene)), and ubiquitination (covalent linkage to protein ubiquitin). For a more detailed controlled vocabulary of PTMs managed by the protein database UniProt, see www.

The term "glycopeptide/glycoprotein" as used herein is a modified peptide/protein having covalently bonded carbohydrates or glycans during or after its synthesis. In certain embodiments, the glycopeptide is obtained from a monoclonal antibody, e.g., a protease digest of a monoclonal antibody.

The term "glycan" as used herein is a compound comprising one or more saccharide units, typically including glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuNAc) (Frank Kjeldsen et al, "analytical chemistry (anal. Chem.) -2003, 75, 2355-2361). The glycan moiety in glycoproteins such as monoclonal antibodies is an important feature for identifying their function or cellular location. For example, a particular monoclonal antibody is modified with a particular glycan moiety.

As used herein, the term "sample" refers to a mixture of molecules of such components within a cell culture medium that is processed according to the methods of the invention, including, for example, isolation, analysis, or profiling. The sample may comprise at least one analyte molecule, e.g. a glycopeptide, such as a glycopeptide obtained from a monoclonal antibody, which is processed according to the methods of the present invention, including e.g. isolation, analysis, extraction, concentration or profiling.

As used herein, the terms "analysis" or "analyzing" are used interchangeably and refer to any of a variety of methods of separating, detecting, isolating, purifying, solubilizing, detecting, and/or characterizing a molecule of interest. Examples include, but are not limited to, solid phase extraction, solid phase microextraction, electrophoresis, mass spectrometry, such as multiplexed targeted Selective Ion Monitoring (SIM) -MS followed by iterative MS2 DDA, ESI-MS, SPE HILIC or MALDI-MS, liquid chromatography, such as high performance, e.g., reverse phase, normal phase or size exclusion, ion-pair liquid chromatography, liquid-liquid extraction, e.g., accelerated fluid extraction, supercritical fluid extraction, microwave assisted extraction, membrane extraction, soxhlet extraction, precipitation, clarification, electrochemical detection, staining, elemental analysis, edman degradation, nuclear magnetic resonance, infrared analysis, flow injection analysis, capillary electrochromatography, ultraviolet detection, and combinations thereof.

As used herein, the term "profiling" refers to any of a variety of analytical methods used in combination to provide a content, composition, or characteristic ratio of a compound, such as a protein.

As used herein, "contacting" includes bringing together at least two substances in a solution or solid phase.

As used herein, "peptide mapping analysis" includes experiments in which proteins are digested, the resulting peptides are then isolated and preferably analyzed using LC-MS. In some exemplary embodiments, peptide mapping analysis may be applied to confirm the primary sequence of a protein biopharmaceutical, in which a protein molecule may first be hydrolyzed using a hydrolyzing agent into small peptide fragments, and then the amino acid sequence of each peptide fragment determined by LC-MS analysis, taking into account the predicted sequence of the cDNA and the specificity of the protease used. Data from peptide mapping analysis can also be used to identify and quantify post-translational modifications, confirm disulfide linkages, and even detect amino acid substitution events that are present at very low levels (< 0.1%) (Zeck et al, journal of public science libraries (PloS one), 2012,7, e 40328). During peptide mapping analysis of protein biopharmaceuticals, LC-MS can typically be performed in combination with Ultraviolet (UV) detection to generate so-called UV fingerprints, which can be used alone as identification assays during Quality Control (QC) and drug release.

As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds of a protein. There are several methods for digesting the proteins in the sample using suitable hydrolytic agents, such as enzymatic digestion or non-enzymatic digestion. As used herein, the term "hydrolyzing agent" refers to any one or combination of a number of different agents capable of performing protein digestion. Non-limiting examples of hydrolytic agents that can be enzymatically digested include trypsin, the intracellular protease Arg-C, the intracellular protease Asp-N, the intracellular protease Glu-C, the outer membrane protease T (OmpT), the immunoglobulin degrading enzyme of Streptococcus pyogenes (IdeS), chymotrypsin, pepsin, thermolysin, papain, pronase and proteases from Aspergillus oryzae (Aspergillus Saitoi). Non-limiting examples of hydrolytic agents that can be subjected to non-enzymatic digestion include the use of high temperature, microwaves, ultrasound, high pressure, infrared, solvents (non-limiting examples are ethanol and acetonitrile), immobilized enzyme digestion (IMER), magnetic particle immobilized enzymes, and immobilized on-chip enzymes. For a recent review of available techniques for discussing protein digestion, see Switazar et al, "protein digestion: a summary of Available technologies and Recent Developments (Protein diagnostics: an Overview of the Available technologies and Recent Developments) (J.proteomics Research) 2013, 12, 1067-1077. One or a combination of hydrolytic agents can cleave peptide bonds in proteins or polypeptides in a sequence-specific manner, generating a predictable collection of shorter peptides.

There are several methods available for digesting proteins. One widely accepted method of digesting proteins in a sample involves the use of proteases. Many proteases are available, and each of them has its own characteristics in terms of specificity, efficiency and optimal digestion conditions. Proteases refer to both endopeptidases and exopeptidases, classified according to their ability to cleave at the non-terminal or terminal amino acid of a peptide. Alternatively, proteases also refer to six different classes-aspartic, glutamic and metalloproteases, cysteine, serine and threonine proteases, as classified according to the catalytic mechanism. The terms "protease" and "peptidase" are used interchangeably and refer to enzymes that hydrolyze peptide bonds. Proteases can also be divided into specific and non-specific proteases. As used herein, the term "specific protease" refers to a protease that has the ability to cleave a peptide substrate at a particular amino acid side chain of the peptide. As used herein, the term "non-specific protease" refers to a protease with reduced ability to cleave a peptide substrate at a specific amino acid side chain of the peptide. Cleavage preference can be determined based on the ratio of the number of specific amino acids as cleavage sites to the total number of amino acids cleaved in the protein sequence.

The protein may optionally be prepared prior to characterization. In some exemplary embodiments, the protein preparation comprises a protein digestion step. In some specific exemplary embodiments, protein preparation includes a protein digestion step, wherein the protein digestion may be performed using trypsin.

In some exemplary embodiments, protein preparation may include the steps of denaturing the protein, reducing the protein, buffering the protein, and/or desalting the sample prior to the protein digestion step. These steps may be accomplished in any suitable manner as desired.

To provide characterization of different protein attributes using peptide mapping analysis or complete mass analysis, a variety of LC-MS based assays can be performed.

As used herein, the term "liquid chromatography" refers to a process in which a chemical mixture carried by a liquid can be separated into components due to the differential distribution of chemical entities as they flow around or over a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and hydrophobic chromatography.

As used herein, the term "mass spectrometer" refers to a device capable of detecting a particular molecular species and accurately measuring its mass. The term may be meant to include any molecular detector into which a polypeptide or peptide may be eluted for detection and/or characterization. The mass spectrometer consists of three main parts: an ion source, a mass analyzer, and a detector. The ion source functions to generate gas phase ions. The analyte atoms, molecules or clusters can be transferred to the gas phase and simultaneously ionized (as in electrospray ionization). The choice of ion source depends on the application.

As used herein, "mass analyzer" refers to a device that can separate species (i.e., atoms, molecules, or clusters) according to the mass of a substance. Non-limiting examples of mass analyzers that can be used are time of flight (TOF), magnetic/electrical sectors, quadrupole mass filters (Q), quadrupole Ion Traps (QIT), orbitrap, fourier Transform Ion Cyclotron Resonance (FTICR), and Accelerator Mass Spectrometry (AMS) techniques.

As used herein, "mass-to-charge ratio" or "m/z" is used to refer to the dimensionless quantity formed by dividing the mass of an ion in unity atomic mass unit by its number of charges (regardless of sign). Generally, the state of charge depends on: ionization methods (such as electrospray ionization, ESI tend to promote multiple ionization, which is not common in MALDI), peptide length (such as longer peptides have more groups where additional protons (basic residues) can be attached), peptide sequence (such as some amino acids (e.g., arg or Lys) ionize more easily than others), instrument settings, solvent pH and solvent composition.

As used herein, the term "tandem mass spectrometry" refers to a technique by which structural information of sample molecules can be obtained by using multiple stages of mass selection and mass separation. A prerequisite is that the sample molecules can be transferred into the gas phase and ionized intact, and that they can be induced to split in some predictable and controllable way after the first mass selection step. Multistage MS/MS or MSn can be performed by first selecting and separating precursor ions (MS 2), fragmenting them, separating primary fragment ions (MS 3), fragmenting them, separating secondary fragments (MS 4), etc., as long as meaningful information is available or the fragment ion signal is detectable. Tandem MS has been successfully performed with various combinations of analyzers. Which analyzers to combine for a particular application can be determined by many different factors such as sensitivity, selectivity and speed, as well as size, cost and availability. The two main categories of tandem MS methods are spatial tandem and temporal tandem, but there are also hybrids where a temporal tandem analyzer is coupled in space or with a spatial tandem analyzer.

A spatial tandem mass spectrometer includes an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. The particular m/z separation function can be designed such that ions are selected in one portion of the instrument, dissociated in the intermediate region, and the product ions are then transported to another analyzer for m/z separation and data acquisition.

In a time tandem mass spectrometer, ions generated in an ion source can be trapped, separated, fragmented, and m/z separated in the same physical device.

As used herein, "targeted mass spectrometry" is a mass spectrometry technique that uses multi-stage tandem mass spectrometry (MSn of n =2 or 3) for ions of a particular mass (m/z) at a particular time. The values of m/z and time are defined in an inclusion list derived from previous analysis.

As used herein, the term "quadrupole-orbitrap mass spectrometer" refers to a hybrid system made by coupling a quadrupole mass spectrometer to an orbitrap mass analyzer. Tandem real-time experiments using a quadrupole-orbitrap hybrid mass spectrometer begin with ejection of all ions from the quadrupole mass spectrometer except for ions within a selected narrow m/z range. Selected ions may be inserted into the orbitrap and fragmented, most often by low energy CID. Fragments within the m/z acceptance range of the trap should remain in the trap and MS-MS spectra can be obtained. Similar hybridization systems can be used for rapid protein sequencing, such as but not limited to QIT-FTICR and Qq-FTICR.

As used herein, the term "de novo sequencing of proteins" refers to a procedure that determines the amino acid sequence of a peptide without relying on information obtained from other sources. Due to the high sensitivity of mass spectrometry, this technique can provide important information that often exceeds the capabilities of conventional sequencing methods.

As used herein, the term "protein sequence coverage" refers to the percentage of a protein sequence that is covered by an identified peptide. Percent coverage can be calculated by dividing the number of amino acids in all found peptides by the total number of amino acids in the entire protein sequence.

As used herein, the term "database" refers to a bioinformatics tool that provides the possibility to search the unexplained MS-MS spectrum for all possible sequences in the database. Non-limiting examples of such tools are Mascot (w.matrix. Com), spectrum Mill (w.chem.agilent. Com), PLGS (w.waters. Com), PEAKS (w.bio.bio.analysis. Com), proteinpilot (down.applied systems. Com// Proteinpilot), phenyx (w.phenyx-ms. Com), sorcer (w.salesearch. Com), OMSSA (w.puche.n.n.n.gov/ssa /), X! Tandem (www.the gpm.org/Tandem /), protein (www.protein.ucsf.edu/Protein/mshome. Htm), byonic (www.protein. Communications.com/products/Byonic), or sequence (fields. Documents.edu/sequence).

General description

As can be appreciated from the above, there is a need for improved methods and systems to improve compound detection and characterization. The disclosed invention meets this need. Disclosed herein are methods for high confidence compound identification by liquid chromatography-mass spectrometry (LC-MC), such as for antibody process development. Embodiments disclosed herein provide methods for robust, high-sensitivity sample characterization by combining multiplexed targeted SIM and iterative MS2 DDA acquisitions.

In some exemplary embodiments, the method includes providing a sample to a liquid chromatography system capable of sample separation to generate sample components; analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list; and performing iterative mass spectrometry Data Dependent Acquisition (DDA) from the inclusion list to identify individual sample components to characterize the sample.

In some embodiments, providing the sample to a liquid chromatography system capable of sample separation to generate the sample component comprises providing the sample to a Reverse Phase Liquid Chromatography (RPLC) system, an ion exchange chromatography system, a size exclusion chromatography system, an affinity chromatography system, a hydrophilic interaction chromatography system, or a hydrophobic chromatography system.

In some embodiments, the liquid chromatography system is an RPLC system. In some specific examples, the RPLC analysis was performed using a Supelco Discovery HS F5-3 column. The column temperature can be maintained at a constant temperature throughout the chromatography run, for example using a commercial column heater. In some embodiments, the column is maintained at a temperature between about 18 ℃ to about 70 ℃, e.g., a temperature of about 30 ℃ to about 60 ℃, about 40 ℃ to about 50 ℃, e.g., about 20 ℃, about 25 ℃, about 30 ℃, about 35 ℃, about 40 ℃, about 45 ℃, about 50 ℃, about 55 ℃, about 60 ℃, about 65 ℃, or about 70 ℃. In some embodiments, the column temperature is about 40 ℃. In some embodiments, the run time may be about 15 to about 240 minutes, such as about 20 to about 70 minutes, about 30 to about 60 minutes, about 40 to about 90 minutes, about 50 minutes to about 100 minutes, about 60 to about 120 minutes, about 50 to about 80 minutes.

In some embodiments, the mobile phase is an aqueous mobile phase. A representative aqueous mobile phase contains 140mM sodium acetate and 10mM ammonium bicarbonate. The UV traces are typically recorded at 215 and 280 nm.

In some exemplary embodiments, the mobile phase used to elute the protein may be a mobile phase compatible with a mass spectrometer.

In some exemplary embodiments, the mobile phase for the liquid chromatography apparatus may comprise water, acetonitrile, trifluoroacetic acid, formic acid, or a combination thereof.

In some exemplary embodiments, the mobile phase may have a flow rate of about 0.1 ml/min to about 0.4 ml/min in the liquid chromatography device. In one aspect, the flow rate of the mobile phase in the liquid chromatography device can be about 0.1 ml/min, about 0.15 ml/min, about 0.20 ml/min, about 0.25 ml/min, about 0.30 ml/min, about 0.35 ml/min, or about 0.4 ml/min.

In some embodiments, analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list comprises utilizing an ion trap or orbitrap mass analyzer. In some embodiments, the ion trap or orbitrap mass analyser is a Thermo Q active HF orbitrap mass spectrometer.

In some embodiments, the sample components are analyzed by multiplexed target-Selective Ion Monitoring (SIM) to generate a list-of-inclusion segmented mass ratio window set, wherein a plurality of segments are included and each segment has a plurality of windows. In some embodiments, at least two, at least three, at least four, at least five, or more segments are used. In some embodiments, three segments are used. In some embodiments, four segments are used.

In some embodiments, each segment includes multiple windows of the same width. In some embodiments, at least 2 or more windows are used, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 windows are used. In some embodiments, each segment includes 10 windows.

In some embodiments, the window width varies between the plurality of segments. For example, the window width in the m/z range of most interest is narrower than the window width of less interest. In some embodiments, segments one and two have narrower window widths than segments three and four. For example, as shown in FIG. 2, an exemplary group may include four segments from 60 to 760.5 m/z: segment 1, 60m/z to 155.5m/z, window width 10Da; segment 2, 155m/z to 250.5m/z, with a window width of 10Da; segment 3, 250m/z to 505.5m/z, with a window width of 25Da; and segments 4, 505.5m/z to 760.5m/z, with a window width of 25Da, wherein each segment comprises 10 windows and a 0.5Da overlap between adjacent windows.

In an embodiment, after generating the inclusion list, the method includes performing iterative MS2 DDA from the inclusion list to identify individual sample components to characterize the sample.

In some exemplary embodiments, the iterative MS DDA utilizes an ion trap or an orbitrap mass analyzer equipped with a segmented quadrupole mass filter.

In some exemplary embodiments, the iterative MS DDA utilizes a commercially available ion trap or Orbitrap mass analyzer equipped with a segmented quadrupole mass filter, such as Thermo Orbitrap ^TM Fusion Lumos mass spectrometer.

In some exemplary embodiments, data is processed after iterating the MS DDA, e.g., by using

In some embodiments, the sample is a cell culture medium, such as a cell culture medium used in fed-batch cell culture, continuous cell culture, or perfusion cell culture.

In some embodiments, the sample cell culture medium is a soy-based cell culture medium.

In some exemplary embodiments, the cell culture medium is used in a recombinant cell-based production system, such as a mammalian system. In some embodiments of the present invention, the,

in some embodiments, the method is used to characterize components and metabolites thereof in cell culture media before or after incubation with recombinant cell-based production systems. In some embodiments, recombinant cell-based production systems are used for protein production. Exemplary proteins include, but are not limited to, antibodies, fusion proteins, recombinant proteins, or combinations thereof.

In some embodiments, the antibody is a bispecific antibody, an antibody fragment, or a multispecific antibody.

In some exemplary embodiments, the antibody is a monoclonal antibody, such as, but not limited to, a monoclonal antibody of isotype IgG1, igG2, igG3, igG4, or mixed isotypes.

In some exemplary embodiments, the protein is a therapeutic protein.

In some exemplary embodiments, the protein may be an immunoglobulin.

In one exemplary embodiment, the protein may be a protein variant.

In one exemplary embodiment, the protein may be a post-translationally modified protein.

In an exemplary embodiment, the post-translationally modified protein may be formed by cleavage, N-terminal extension, protein degradation, acylation at the N-terminus, biotinylation, amidation at the C-terminus, oxidation, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation, alkylation, methylation, adenylation, ADP-ribosylation, covalent cross-linking within or between polypeptide chains, sulfonation, prenylation, vitamin C-dependent modification, vitamin K-dependent modification, glutamation, glycination, glycosylation, deglycosylation, prenylation, lipoylation, phosphopantetheinylation, phosphorylation, sulfation, citrullination, deamidation, formation of disulfide bridges, proteolytic cleavage, ISG acylation, SUMO acylation, or ubiquitination (covalent attachment to protein ubiquitin).

In one exemplary embodiment, post-translationally modified proteins may be formed upon oxidation of the protein.

In another exemplary embodiment, the cell culture medium can include a degradation product, such as a post-translational modification of a therapeutic protein.

In some exemplary embodiments, the protein may be a protein having a pI in the range of about 4.5 to about 9.0. In one aspect, the protein may be a protein having a pI of about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.

In some embodiments, the identified components include, but are not limited to, amino acids, dipeptides, tripeptides, or combinations thereof, having a dynamic range of abundance of at least 2 orders of magnitude, such as at least 3 orders of magnitude, at least 4 orders of magnitude, or greater. In some embodiments, the identified components include one or more of the compounds provided in fig. 7.

In some exemplary embodiments, the one or more compounds/components detected may include one or more product-related impurities. Exemplary product-related impurities can be, but are not limited to, molecular variants, precursors, degradation products, fragmented proteins, digestion products, aggregates, post-translational modifications, or combinations thereof.

In some specific exemplary embodiments, the one or more compounds/components detected may be process-related impurities. Process-related impurities may include impurities derived from manufacturing processes, for example, nucleic acids and host cell proteins, antibiotics, serum, other media components, enzymes, chemical and biochemical treatment reagents, inorganic salts, solvents, carriers, other leachables used in ligand manufacturing processes that may be present or released into the cell culture media.

In some embodiments, the disclosed method is a compound identification method for cell culture media analysis comprising: providing a cell culture medium sample to a liquid chromatography system capable of sample separation to generate sample components; analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list; and performing iterative mass spectrometry Data Dependent Acquisition (DDA) from the inclusion list to identify individual compounds within the cell culture medium. In some examples, the method further comprises, for example, culturing the recombinant protein (e.g., antibody) -producing cell in a cell culture medium, obtaining a sample from the cell culture at a desired time point prior to characterizing the cell culture medium components.

In some embodiments, the method comprises identifying a cell culture medium component by using the disclosed methods, followed by altering one or more culture conditions of the cell culture to reduce the amount of the characteristic compound produced during cell culture of the protein. Exemplary conditions of the cell culture that may be altered include, but are not limited to, temperature, pH, cell density, amino acid concentration, osmotic pressure, growth factor concentration, agitation, partial pressure of gas, surfactants, or combinations thereof.

In some embodiments, the cell that produces a protein such as an antibody is a CHO cell. In other embodiments, the cell is a hybridoma cell.

Examples of the invention

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees celsius, room temperature is about 25 ℃, and pressure is at or near atmospheric.

Example 1: materials and methods

The combined soy hydrolysates were dissolved in Milli-Q water as a base. Standards of stable isotopically labeled compounds are incorporated to final concentrations ranging from nanomolar to hundreds of millimolar. Reversed Phase Liquid Chromatography (RPLC) separation was performed using a Supelco Discovery HS F5-3 column. Multiplexed target-Selective Ion Monitoring (SIM) was performed on a Thermo Q active HF orbitrap mass spectrometer to generate an inclusion list. Iterative MS2 Data Dependent Acquisition (DDA) including lists was performed on a Thermo Orbitrap Fusion Lumos mass spectrometer. A MS1 full scan was performed followed by a conventional MS2 DDA for comparison. Data processing was performed using Compound discover 3.1.

Example 2: multiplexing targeted SIM and iterative MS2 DDA acquisitions increases robust compound identification for cell culture medium analysis.

Multiplexed targeted SIM and iterative MS2 DDA acquisitions were performed using the materials and methods described in example 1. Fig. 1 provides a schematic diagram illustrating two types of MS1 data acquisition according to embodiments disclosed herein: (1) Conventional full scan MS1 acquisition and (2) targeted SIM MS1 acquisition. Targeting SIM MS1 acquisition relies on size-based molecular classification. By comparison, the full scan MS1 detects a specific number of molecules regardless of their size. As shown, targeted SIM MS1 acquisition allows lower abundance species to be detected when only a specific number of molecules of each size are detected, thereby increasing the confidence of the detection. Targeting the SIM MS1 includes optimizing the injection time and isolation window.

In particular, FIG. 2 provides a schematic diagram depicting an exemplary adjustable window selection scheme comprising four segments ranging from 60 to 760.5 m/z: segment 1, 60m/z to 155.5m/z, window width 10Da; segment 2, 155m/z to 250.5m/z, with a window width of 10Da; segment 3, 250m/z to 505.5m/z, with a window width of 25Da; and segment 4, 505.5m/z to 760.5m/z, with a window width of 25Da. Each segment includes 10 windows and a 0.5Da overlap between adjacent windows. The width of the window varies between segments, however, it does not vary within a particular segment. The region of particular interest has a smaller window width. Fig. 3 shows MS1 spectra acquired after a conventional full scan MS1 acquisition (top panel) or an adjustable window SIM MS1 acquisition (bottom panel) according to embodiments disclosed herein. Multiplexing the targeted SIM provides a clearer background and higher intensity for several incorporated stable isotope labeled compounds compared to MS1 full scan. For example, 10nM of stable isotope labeled histidine was only observed by the multiplexed targeted SIM method.

MS2 DDA is performed after MS1 full scan or multiplexing the targeted SIM. Fig. 4 shows a schematic diagram illustrating two types of MS2 DDA, according to embodiments disclosed herein: (1) conventional DDA MS2 acquisition and (2) iterative DDA MS2 platform.

Figure 5 shows the results of iterative MS2 data acquisition of selecting and fragmenting more species than conventional DDA MS2 acquisition of only selecting and fragmenting abundant species. Fig. 6 shows a schematic diagram of a conventional method of comparing a characterization sample and using a combination of an adjustable window SIM acquisition and an iterative MS2 acquisition, according to embodiments disclosed herein. As shown in fig. 7, the combination of the adjustable window SIM acquisition and the iterative MS2 acquisition identified a variety of components of high complexity in the soy hydrolysate sample, including amino acids, dipeptides, tripeptides, etc., with a dynamic range of abundance of at least 4 orders of magnitude. Fig. 8 shows a graph illustrating the intensity of an iterative MS2 acquisition. The top row represents the number of entries on the filter exclusion list, while the bottom row is the number of entries on the filter inclusion list, each entry associated with an iterated sample injection. As shown, as the iterative feed increases, the exclusion list of species increases while the inclusion list decreases, which in turn allows for the detection of lower abundance species. In the iterative MS2 DDA, background ions in the blank run were successfully excluded during the sample run. MS2 scans were triggered on ions of much lower abundance by multiple iterations of the sample run. Rapid and high confidence component identification is achieved by searching internal libraries (in-house library) containing accurate mass, retention time and fragmentation spectra, among other online databases. It is contemplated that the disclosed methods may be used to analyze cell metabolites, for example, for CHO cell metabolite analysis during cell culture development.

In summary, the disclosed methods provide a more robust, sensitive method for characterizing samples such as cell culture media and individual components and metabolites therein, which can be used to improve antibody process development.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method of characterizing a sample, comprising:

providing the sample to a liquid chromatography system capable of sample separation to generate sample components;

analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list; and

performing iterative mass spectrometry Data Dependent Acquisition (DDA) from the inclusion list to identify individual sample components.

2. The method of claim 1, wherein the liquid chromatography system is a Reverse Phase Liquid Chromatography (RPLC) system.

3. The method of claim 1 or claim 2, wherein analyzing the fragmented sample by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list comprises utilizing an ion trap or orbitrap mass analyzer.

4. The method of any of claims 1-3, wherein performing iterative mass spectrometry (DDA) comprises utilizing an ion trap or an orbitrap mass analyzer equipped with a segmented quadrupole mass filter.

5. The method of any one of claims 1 to 4, wherein analyzing sample components by multiplexed target-Selective Ion Monitoring (SIM) to generate a list comprises a segmented mass ratio window set comprising a plurality of segments and each segment having a plurality of windows.

6. The method of claim 5, wherein the plurality of segments is three segments.

7. The method of claim 5, wherein the plurality of segments is four segments.

8. The method of any of claims 5 to 7, wherein the plurality of windows is 10 windows.

9. The method of any of claims 5 to 8, wherein each window within a segment has the same window width.

10. The method of any one of claims 1 to 9, wherein the sample is a cell culture medium.

11. The method of claim 9, wherein the method is used to characterize components and metabolites thereof in the cell culture medium.

12. The method of claim 10 or claim 11, wherein the cell culture medium is a soy-based cell culture medium.

13. The method of claim 10 or 11, wherein the cell culture medium is used in a recombinant cell-based production system.

14. The method according to any one of claims 1 to 10, wherein the method is used for characterizing components in the cell culture medium and metabolites thereof after incubation with a recombinant cell based production system.

15. The method of claim 14, wherein the recombinant cell-based production system is a mammalian system.

16. The method of claim 14, wherein the recombinant cell-based production system is for protein production.

17. The method of claim 16, wherein the protein is an antibody, a fusion protein, a recombinant protein, or a combination thereof.

18. The method of claim 17, wherein the antibody is a monoclonal antibody.

19. The method of claim 18, wherein the monoclonal antibody is of isotype IgG1, igG2, igG3, igG4, or mixed isotypes.

20. A method of compound identification for cell culture media analysis comprising:

providing a cell culture medium sample to a liquid chromatography system capable of sample separation to generate sample components;

performing iterative mass spectrometry Data Dependent Acquisition (DDA) from the inclusion list to identify individual compounds in the cell culture medium.

21. The method of claim 20, wherein the liquid chromatography system is a Reverse Phase Liquid Chromatography (RPLC) system.

22. The method of claim 20 or claim 21, wherein analyzing the fragmented sample by multiplexed target-Selective Ion Monitoring (SIM) to generate an inclusion list comprises using an ion trap or orbitrap mass analyser.

23. The method of any of claims 20 to 22, wherein performing iterative mass spectrometry (DDA) comprises utilizing an ion trap or an orbitrap mass analyser equipped with a segmented quadrupole mass filter.

24. The method of any one of claims 20 to 23, wherein analyzing sample components by multiplexed target Selective Ion Monitoring (SIM) to generate a list comprises a segmented mass ratio window set comprising a plurality of segments and each segment having a plurality of windows.

25. The method of claim 24, wherein the plurality of segments is three segments.

26. The method of claim 24, wherein the plurality of segments is four segments.

27. The method of any one of claims 24 to 26, wherein the plurality of windows is 10 windows.

28. The method of any one of claims 24 to 27, wherein each window in a segment has the same window width.

29. The method of any one of claims 20 to 28, wherein the cell culture medium is a soy-based cell culture medium.

30. The method of any one of claims 20-29, wherein the cell culture medium is used in a recombinant cell-based production system.

31. The method of any one of claims 20 to 30, wherein the cell culture medium sample is a cell culture medium sample obtained after incubation with a recombinant cell-based production system.

32. The method of claim 31 or claim 32, wherein the recombinant cell-based production system is a mammalian system.

33. The method of claim 31 or claim 32, wherein the recombinant cell-based production system is for protein production.

34. The method of claim 33, wherein the protein is an antibody, a fusion protein, a recombinant protein, or a combination thereof.

35. The method of claim 34, wherein the antibody is a monoclonal antibody.

36. The method of claim 35, wherein the monoclonal antibody is of isotype IgG1, igG2, igG3, igG4 or mixed isotypes.